1. Structural Subtypes and Biological Activities of PARP
Poly(ADP-ribose)polymerases (PARP), which exist in eukaryotic cells and catalyze the polymerization of ADP-ribose, include numerous family members. PARP1 is the earliest-discovered ribozyme in cell that can catalyze ribosylation of poly ADP, and later, other subtypes, such as PARP2, PARP3, PARP4 (VPARP), PARP5a (tankyrase 1), PARP5b (tankyrase 2), PARP7 (TiPARP) and sPARP1, were also separated subsequently. At present, 18 subtypes having potential PARP activity have been determined according to the structure of catalytic domain of PARP1, in which PARP1 has a relatively complete structure. PARP1 contains three main domains, a DNA-binding domain (DBD) at N-terminal, an automodification domain (AMD) and a catalytic domain at C-terminal. The DBD comprises two zinc-finger (ZnF) domains and DNA strand break sensitive element (NLS), and zinc-finger (ZnF) domain will bind to the damaged parts of DNA strand and repair such parts by receiving signals of DNA strand breaks through NLS. In the PARP family, the homology between PARP-2 and PARP1 is the highest which is 69%. Therefore, the currently reported PARP1 inhibitors generally have compatible activity on PARP2 as well.
2. PARP and Diseases
Of the known PARP related functions, PARP1 plays dominantly. These particularly include: 1) repairing DNA and maintaining genome stability; 2) regulating both transcription level and expression of related proteins; 3) affecting replication and differentiation, and maintaining telomere length; 4) regulating cell death and removing damaged cells. Therefore, the DNA repairing mechanism mediated by PARP1 may be inhibited and the damage of radiotherapy and chemotherapy on tumor cell DNA may be increased by inhibiting the PARP1 activity, thereby having a therapeutic effect on tumor.
Although PARP has DNA repair function, but when DNA damage is excessive and difficult to be repaired, PARP will be over-activated and tend to have a “suicide mechanism” leading to over-consumption of the substrate nicotinamide adenine dinucleotide (NAD+) and ATP, depletion of cell energy, and cell necrosis, and ultimately organ tissue injury that is one of the pathogenesis of brain injury and neurodegenerative diseases. It is shown that PARP1 inhibitors exhibit therapeutical effects in animal models of cerebral ischemic injury, shock, Alzheimer and Parkinsonian diseases. Therefore, PARP1 inhibitors have a therapeutic effect for various ischemic and neurodegenerative diseases.
3. PARP Inhibitors
It has been reported by Armin et. al. that the catalytic active sites of PARP1 can be roughly divided into two domains, donor domain and acceptor domain, both using PARP substrate NAD+ as a scaffold. Acceptor domain binds to ADP of poly adenosine ribose diphosphate chains. Donor domain binds to NAD+, and is further divided into three sub-binding domains: nicotinamide-ribose binding site (NI site), phosphate binding site (PH site), and adenosine-ribose binding site (AD site). Most of the reported PARP inhibitors interact with the NI site of PARP and competitively inhibit NAD+, therefore, their structures are similar to that of nicotinamide. For example, AZD2281 (olaparib/KU-59436) developed by AstraZeneca is an oral small molecule PARP inhibitor, has showed promising therapeutical effects in treating ovarian cancer, breast cancer and solid tumor in combination with drugs such as cisplatin, carboplatin, paclitaxel and so on, and is currently in phase II clinical stage.

However, the in vivo action time and half-life time (<1 hours) of compound AZD2281 are relatively short, and its bioavailability is low (<15%), which may limit its further development. There are many reasons leading to these shortcomings, and the cyclic tertiary amine of its chemical structure is one of the main reasons that cause the metabolic instability. The cyclic tertiary amine can form oxidation product I or imine intermediate II by oxidase or P450 metabolic enzymes (as shown in the above figure), thus producing a series of oxidative products, including metabolites from N-dealkylation, ring hydroxylation, alpha-carbonylation, N-oxidation, ring opening and so on. All these metabolic products can result in metabolic inactivation of the drug, and even produce toxicity. For example, the cyclic tertiary amine fragment can be metabolized into MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydrogen pyridine), or phencyclidine (hallucinogenic drugs) and so on through imine intermediate, thereby producing central nervous system toxicity. Meanwhile, AZD2281 has relatively low selectivity within the PARP family, especially to telomerase Tankyrase 1 and Tankyrase 2, which may cause clinical safety concerns.
Therefore, on the basis of a comprehensive analysis on the binding characteristics of the crystal structure of PARP1 with small molecule compounds such as AZD2281, in the present invention, we designed a series of new PARP1 inhibitors by maintaining the key hydrogen bonding sites which will influence activity, i.e. amide segment, and modifying the hydrophobic part, mainly through 1) introducing the piperazinotriazole system with substituents to increase the steric hindrance of tertiary amine, or substituting the metabolic sites to reduce oxidative metabolism ability of compounds under the action of cytochrome P450 enzyme system in vivo, thereby increasing the stability in vivo of molecules and reducing the likelihood of generating toxic metabolites; 2) introducing one or more substituents on the piperazine ring to increase selectivity over telomerase Tankyrase 1 and Tankyrase 2, thereby improving the safety of compounds as PARP1 inhibitors in treating diseases. Therefore, a series of piperazinotriazole compounds containing one or more substituents were developed as novel highly selective PARP1 inhibitors with potential use in treating various ischemic diseases, neurodegenerative disorders and cancers.